The present invention relates to peptides derived from the env gene of FIV (feline immunodeficiency virus), as well as to their immunoprotective uses (prevention and treatment of feline immunodeficiency).
Feline immunodeficiency is due to a lentivirus, feline immunodeficiency virus (FIV), which has a genetic structure similar to that of the lentiviruses of primates (HIV and SIV).
A certain number of fragments have been selected and have allowed the development of sensitive and specific tests for detecting seropositive animals, as described in European patent applications No. 0 564 477 of Nov. 20, 1991, No. 0 577 458 of Jun. 16, 1993 and French patent application No. 94/07062 of Jun. 9, 1994, in the name of the Applicant, and are mostly derived from the Env protein of FIV, comprising 854 amino acids, the sequence of which is described in European patent application No. 0 577 458.
The Env protein gives, after cleavage, 2 glyco-protein fragments referred to as SU (surface glycoprotein) and TM (transmembrane glycoprotein), in which nine domains, comprising continuous B epitopes of the envelope glycoproteins of the FIVs and recognized during the natural infection (Pancino G. et al., J. Virol, 1993, 67, 664-672), have been defined.
Among these nine domains, five (SU1-SU5) of them are located on the surface glycoprotein and four of them (TM1-TM4) are located on the transmembrane glycoprotein.
More specifically, the positions of the domains TM1-TM4 in the TM protein are as follows:
the TM1 domain (51 amino acids) corresponds to positions 595-647 of the Env protein of FIV,
the TM2 domain (31 amino acids) corresponds to positions 681-711 of the Env protein of FIBV; this domain contains an epitope including the sequence: Cys697-Asn-Gln-Asn-Gln-Phe-Phe-Cys-Lys705 (peptide referred to as P237),
the TM3 domain (45 amino acids) corresponds to positions 744-788 of the Env protein of FIV, and
the TM4 domain (29 amino acids corresponds to positions 826-854 of the Env protein of FIV.
Whereas in the field of detection, a set of reagents is now available to detect FIV, in the immunoprotection field, the situation is more complex.
The TM2 peptide, for example, can induce the formation of facilitating antibodies, which have an effect contrary to that which is desired, i.e. an effect of amplifying the viral infection instead of having a protective effect.
In particular, it is observed that vaccination with preparations of FIV envelope entails a clinical aggravation or acceleration of the viral infection.
Although it is usually recognised that the function of antibodies is conventionally estimated by tests of neutralization of the viral infectivity of cells in culture, the results observed have shown that most of the neutralizing antibodies are directed against the envelope glycoproteins, but that a vaccination with the lent iviral envelope, despite the induction of neutralizing antibodies, does not make it possible to obtain a suitable protection with respect to experimental infections (Johnson R. P., Curr. Opinion in Immunol., 1996, 8, 554-560).
In particular, after immunization with the FIV envelope glycoprotein and a virulence test, either a decrease in the viral load (Hosie M. J. et al., Vaccine, 1996, 14, 405-411) or an aggravation of the primary infection (Siebelink K. H. et al., J. Virol., 1995, 69, 3, 3704-3711) was observed in cats. In the latter study (Siebelink K. H. et al., 1995, mentioned above), the aggravation of the infection with FIV is also observed in animals subjected to a passive transfer of plasma from immunized cats; such results suggest the possibility that it is the antibodies which are inducing the increase in the Infection observed IBM vivo.
The possibility of the immune response directed against the FIV envelope being deleterious to the host was confirmed by the Inventors, in a vaccination experiment with the env gene, which led to an acceleration of the infection.
However, both the existence of a causal link between the aggravation of the infection and the presence of antibodies, and the existence of a link between the degree of protection obtained and the presence of antibodies, are difficult to establish and show the complexity encountered for developing compositions that are effectively protective with respect to lentiviruses and more particularly FIV. The reason for this is that he presence of facilitating antibodies in the serum of patients infected with HIV-1 has been correlated, in certain studies, with the progression of the disease (Fust G. et al., AIDS, 1994, 8, 603-609; Hornsy J. et al., J. Virol., 1990, 64, 1437-1440).
It is in particular strongly possible that the antibodies which increase the lentiviral infectivity can also reduce the degree of protective immunity obtained during the natural infection, after vaccination with subunits of envelope protein.
Although the induction of antibodies which neutralise the viral infection in vitro was considered hitherto as a good indicator for selecting sequences capable of inducing a protective immunity, the abovementioned results show that there is in fact no neutralizing activity/protection correlation (Matteuci D. et al., J. Virol. , 1996, 70, 617-622)
In addition, the conditions for measuring the activity and the choice of viruses on which the tests are carried out intervene in the interpretation of the results.
Particularly influential parameters which may be mentioned are: the cell substrate used to measure the residual infectivity and the passages of the virus, the use of primary isolates relative to the viruses adapted in the laboratory.
The selection of monoclonal antibodies with strong neutralizing power and directed against conformations epitopes has suggested that discontinuous epitpes are important of the protection. However, the identification and synthesis of mimotopes for vaccinal use are particularly difficult to achieve.
Peptides, representing continuous epitopes, are reagents that are entirely suitable for vaccine purposes. The continuous neutralizing epitope of HIV-1 which has beer, most extensively studied is the main neutralizing domain of the surface glycoprotein, the V3 domain (Goudsmit J. et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 4478-4482; Palker T. J. et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 1932-1936; Rusche J. R. et al., Proc. Natl. Acad. Sci. USA, 988, 85, 3198-3202). However, this region is hypervariable and the neutralization is essentially limited to the homologous virus.
A peptide derived from the V3 region of FIV, which induces neutralizing antibodies, induced no antiviral protection in immunized cats (Lombardi, J. Virol, 1994, 68, 8374-8379). This clearly shows that the in vitro neutralization does not directly signify protective capacity.
Given the complexity of the reactivity of the immune system with respect to lentiviral proteins and the fact that not all the neutralizing antibodies systematically have protective activity, the Inventors set themselves the aim of using synthetic peptides such as those described in European patent application No. 0 577 458 to prepare a medicinal product capable of providing a certain degree of protection against an infection with FIV and of selecting novel synthetic peptides capable of inducing a certain degree of protection against a virulence test with a primary strain of FIV; such a protection is observed by the reduction or suppression of the viral load during the acute infection.
A subject of the present invention is the use of peptides selected from the group consisting of:
peptides containing from 12 to 19 amino acids and whose sequence is contained in SEQ ID NO:1 below: Lys-Lys-Gly-Leu-Gln-Gln-Leu-Gln-Glu-Trp-Glu-Asp-Trp-Val-Gly-Trp-Ile-Gly-Asn (SEQ ID NO: 1) and
peptides containing not more than 50 amino acids comprising the said sequence SEQ ID NO:1, to prepare a medicinal product capable of inducing a certain degree of protection against an infection with. FIV.
A subject of the present invention is also peptides capable of inducing a certain degree of protection (preventive or therapeutic) against an infection with FIV, characterized in that they are selected from the group consisting of peptides containing from 12 to 19 amino acids and whose sequence is contained in the sequence SEQ ID NO:1.
The invention includes the analogues of the said peptides in which certain amino acids are substituted, deleted or added, the peptides obtained inducing the same degree of protection with respect to an infection with FIV as that obtained with the peptides as defined above.
Surprisingly, a decrease or an elimination of the viral load is observed during an acute infection, only after immunization with the peptide of SEQ ID NO:1, a fragment thereof comprising at least 12 amino acids or a peptide containing the said sequence SEQ ID NO:1.
The 19-amino-acid peptide of SEQ ID NO:1, also referred to as TM3-19, is a fragment of the peptide corresponding to the TM3 domain.
A systematic analysis of the functional activity of antibodies directed against the various abovementioned domains (SU1-SU5 and TM1-TM4) allowed the identification of only a single domain, SU2, located in the third variable region, as being capable of inducing antibodies wich neutralize the viral infectivity of cells in culture (de Rcnde A. et al., Virology, 1994, 198, 257-264; Lombardi S. et al., J. Virol., 1993, 67, 4742-4749; Richardson J. et al., J. Gen. Virol., 1996, 77, 759-771).
Three domains (SU2, SU5 and TM3-19), recognized in the majority of infected cats, were selected by he Inventors as xe2x80x9ccandidatesxe2x80x9d as inmunogens. However, although the SU2 domain induces the production of neutralizing antibodies, only the fragment containing the sequence SEQ ID NO:1 is effectively protective with respect to the infection.
Surprisingly, the sequence TM3-19 or sequences of less than 50 amino acids containing it, has protective activity (both preventive and curative), although it does not Induce the production of neutralizing antibodies.
A subject of the present invention is also an immunoprotective and/or vaccinal composition against an infect on with FIV, characterized in that it consists essentially of at least one peptide containing from 12 to 19 amino acids as defined above, optionally combined with another peptide which snows immunoreactivity with respect to anti-FIV antibodies (other viral subunits and/or complete envelope glycoprotein) and which is capable of inducing a certain degree of protection against an infection with FIV, and at least one pharmaceutically acceptable vehicle.
A subject of he present invention is also an immunoprotective composition, as defined above, for its use in the prevention (vaccine) or treatment of injections with FIV.
The evaluation of the viral load of FIV and/or monitoring of the efficacy of the vaccination or of the treatment in a biological sample can be carried out by a method which comprises:
(a) extraction of the viral RNA from a biological sample to be analysed;
(b) preparation of a range of samples each containing a different number of a competitive RNA, obtained from a conserved region of the gag gene, into which at least one modification has been introduced;
(c) mixing of the RNA extract obtained in (a) with each sample of the range obtained in (b), separately
(d) reverse transcription of the RNAs present in the various mixtures obtained in (c), to give the corresponding cDNA;
(e) amplification of the cDNAs obtained in (d), using primers selected from the group consisting of the sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10;
(f) separation of the amplification products, and
(g) detection of the number of copies of initial viral RNA by suitable quantitative analysis, such as comparative densitometry and establishment of the ratio: density of products from the competitive RNA/density of products from the wild-type RNA present in the initial sample, as a function of the number of copies of competitive RNA added to each mixture.
The competitive RNA according to step (b) is obtained, when the said modification is a deletion, by:
(i) amplification of the said conserved region using primers of sequence SEQ ID NO:4 and SEQ ID NO:5,
(ii) execution of the deletion by mutagenesis via PCR of the 3xe2x80x2 portion of the product obtained in (i), using primers of sequence SEQ ID NO:6 and SEQ ID NO:5,
(iii) replacement of the terminal 3xe2x80x2 portion of the product obtained in (i) with the fragment bearing the deletion obtained in (ii), and
(iv) production of the said competitive RNA using the final product obtained in (iii) as matrix.